This invention relates to compounds, compositions and methods useful for the detection of analytes by complementation of polypeptide fragments of .beta.-galactosidase. Specifically, the invention relates to cross-linking agents, formation of an intramolecular cross-link in enzyme donor polypeptides of .beta.-galactosidase, and the use of such compositions in the detection and quantitation of analytes in samples.
In the past, various synthetic and natural antigenic polypeptides and polypeptide fragments have been conjugated to high molecular weight protein carriers such as latex functionalized SEPHAROSE (Pharmacia, Inc.), tetanus toxoid, keyhole limpet hemocyanin, agarose and cellulose to detectable labels such as fluorophores, and to chemotherapeutic agents using bifunctional cross-linking agents. U.S. Pat. No. 4,493,795 and PCT publication WO 90/05749 (published May 31, 1990) are exemplary. Such cross-linking agents have also been used to attach bioactive or cytotoxic agents, dyes, radioactive compounds and the like to antibody molecules. U.S. Pat. No. 4,671,958 is exemplary. Antibodies have been linked together using such agents. See Chen, Res. Virol. 141:337-42 (1990). Cross-linking agents have also found use for modifying bioactive and therapeutically useful polypeptides by conjugation with polymers such as polyethylene glycol to enhance pharmacokinetic properties. U.S. Pat. Nos. 5,166,322, 4,179,337 and 4,766,106 are exemplary.
.beta.-Galactosidase is a tetrameric protein with a monomer molecular weight of approximately 116,000 Daltons. The monomer is composed of 1023 amino acids. Intracistronic complementation is the known phenomenon whereby individually inactive peptide fragments of the enzyme spontaneously associate to form an active .beta.-galactosidase protein. Among the first .beta.-galactosidase complementation pairs investigated in depth was the M15/CNBr2 system described by Langley and Zabin, Biochemistry 15:4866 (1976). M15 is a deletion mutant of .beta.-galactosidase lacking amino acids 11-41. The CNBr2 peptide consists of amino acids 3-92 of .beta.-galactosidase and is prepared from cyanogen bromide cleavage of the intact enzyme. When M15 and CNBr2, which are individually inactive, are incubated together under appropriate conditions, the two peptides complement or associate with eachother to form fully active, tetrameric .beta.-galactosidase. In this system, CNBr2, the N-terminal peptide, is referred to as the .alpha.-enzyme donor. M15, which has the N-terminal deletion, is referred to as the .alpha.-enzyme acceptor. The general phenomenon which uses the reassociation of the domains of .beta.-galactosidase to form active .beta.-galactosidase from inactive fragments is referred to as complementation. Other combinations of .alpha.-enzyme donors and .alpha.-enzyme acceptors have been described. See Zabin, Mol. and Cellular Biochem 49:84 (1982). Each is a variant derived from the natural .beta.-galactosidase sequence.
Complementation of a C-terminal peptide and corresponding C-terminal deletion protein has also been described. An example of this phenomenon, known as omega-complementation, is X-90, a .beta.-galactosidase deletion variant lacking 10 amino acids at the C-terminus and CNBr24, a peptide comprising amino acids 990-1021 of .beta.-galactosidase. As in the case of .alpha.-complementation, .omega.-enzyme donor polypeptides and .omega.-enzyme acceptor proteins are inactive but reassociate to form enzymatically active tetramer. See Welphy, Biochem. Biophys. Res. Comm. 93:223 (1980).
.beta.-Galactosidase complementation activity has been exploited to produce sensitive quantitative assays for both high and low molecular weight analytes. U.S. Pat. Nos. 5,362,625 and 4,708,929 disclose, inter alia, a variety of enzyme donor and enzyme acceptor polypeptide compositions for use in antibody and receptor binding assays. The enzyme donors and enzyme acceptors are generated by means of recombinant DNA or polypeptide synthesis techniques familiar to skilled artisans.
These approaches allow great flexibility and control over the design of enzyme donor and enzyme acceptor molecules. The use of genetic engineering techniques allows the sequence and length of the enzyme donor and enzyme acceptor polypeptides to be modified to maximize assay performance and reagent stability. Enzyme donors optimized for chemical coupling to analyte and enzyme donors genetically fused to analyte peptides or proteins have been described, and immunoassays using these compositions are commercially available. See Henderson, Clin. Chem. 32:1637 (1986); Khanna, Amer. Clin. Lab 8:14 (1989) and Coty, J. Clin. Immunoassay 17:144 (1994).
One problem not addressed by the art in this area involves the reduction of background interference in these complementation assays. Because the enzyme donor and enzyme acceptor molecules spontaneously combine to form active enzyme, antibody or receptor binding to the unmodified enzyme donor or enzyme acceptor fragments has been relied upon in the past to inhibit such undesirable complementation. This approach has not fully succeeded.